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- PDB-6uho: Crystal Structure of C148 mGFP-cDNA-2 -

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Basic information

Entry
Database: PDB / ID: 6uho
TitleCrystal Structure of C148 mGFP-cDNA-2
ComponentsC148 mGFP-cDNA-2
KeywordsFLUORESCENT PROTEIN / green fluorescent protein / beta-barrel / DNA / protein-DNA conjugate
Function / homologyGreen fluorescent protein, GFP / Green fluorescent protein-related / Green fluorescent protein / Green fluorescent protein / bioluminescence / generation of precursor metabolites and energy / Unknown ligand / Green fluorescent protein
Function and homology information
Biological speciesAequorea victoria (jellyfish)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.95 Å
AuthorsWinegar, P.W. / Hayes, O.G. / McMillan, J.R. / Figg, C.A. / Focia, P.J. / Mirkin, C.A.
Funding support United States, 2items
OrganizationGrant numberCountry
Department of Defense (DOD, United States)N00014-15-1-0043 United States
Department of Defense (DOD, United States)FA9550-17-1-0348 United States
CitationJournal: Chem / Year: 2020
Title: DNA-Directed Protein Packing within Single Crystals.
Authors: Winegar, P.H. / Hayes, O.G. / McMillan, J.R. / Figg, C.A. / Focia, P.J. / Mirkin, C.A.
History
DepositionSep 27, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 18, 2020Provider: repository / Type: Initial release
Revision 1.1Mar 24, 2021Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Oct 11, 2023Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession
Revision 1.3Nov 15, 2023Group: Data collection / Category: chem_comp_atom / chem_comp_bond / Item: _chem_comp_atom.atom_id / _chem_comp_bond.atom_id_2
Revision 1.4Nov 20, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature / Item: _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: C148 mGFP-cDNA-2
B: C148 mGFP-cDNA-2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)62,1424
Polymers62,0782
Non-polymers642
Water5,711317
1
A: C148 mGFP-cDNA-2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)31,0712
Polymers31,0391
Non-polymers321
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
2
B: C148 mGFP-cDNA-2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)31,0712
Polymers31,0391
Non-polymers321
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)64.710, 52.220, 86.440
Angle α, β, γ (deg.)90.000, 94.230, 90.000
Int Tables number4
Space group name H-MP1211

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Components

#1: Protein C148 mGFP-cDNA-2


Mass: 31038.908 Da / Num. of mol.: 2 / Mutation: S148C
Source method: isolated from a genetically manipulated source
Details: This mutant of EGFP has a single surface cysteine at residue 148 (counting from M37 and counting CRO as 3 residues) and an N-terminal his-tag.
Source: (gene. exp.) Aequorea victoria (jellyfish) / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P42212*PLUS
#2: Chemical ChemComp-UNL / UNKNOWN LIGAND


Mass: 32.065 Da / Num. of mol.: 2 / Source method: obtained synthetically
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 317 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.35 Å3/Da / Density % sol: 47.58 % / Description: Crystal was a thin green plate
Crystal growTemperature: 295 K / Method: vapor diffusion, sitting drop
Details: 1 microliter C148 mGFP-cDNA-2 (5 mg/mL (protein concentration) in 10 mM Tris Buffer pH 7.4, 137 mM NaCl) + 1 microliter crystallization condition (0.15 M potassium bromide, 30% (w/v) PEG MME ...Details: 1 microliter C148 mGFP-cDNA-2 (5 mg/mL (protein concentration) in 10 mM Tris Buffer pH 7.4, 137 mM NaCl) + 1 microliter crystallization condition (0.15 M potassium bromide, 30% (w/v) PEG MME 2000) in a sitting drop with a 70 microliter reservoir (0.15 M potassium bromide, 30% (w/v) PEG MME 2000)

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 21-ID-F / Wavelength: 0.97872 Å
DetectorType: RAYONIX MX-300 / Detector: CCD / Date: Apr 22, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97872 Å / Relative weight: 1
ReflectionResolution: 1.95→86.204 Å / Num. all: 41971 / Num. obs: 41971 / % possible obs: 99.2 % / Redundancy: 4.3 % / Rpim(I) all: 0.051 / Rrim(I) all: 0.107 / Rsym value: 0.094 / Net I/av σ(I): 4.3 / Net I/σ(I): 9.4 / Num. measured all: 179992
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. unique obsRpim(I) allRrim(I) allRsym value% possible all
1.95-2.064.30.4063.361330.2220.4640.406100
2.06-2.184.30.2694.858230.1460.3070.269100
2.18-2.3340.2875.551040.170.3350.28794
2.33-2.524.40.1418.751110.0760.1610.141100
2.52-2.764.40.09510.946740.0510.1080.095100
2.76-3.084.40.0741342920.0390.0840.074100
3.08-3.564.40.0714.937190.0370.0790.07100
3.56-4.364.30.06415.432140.0340.0730.06499.8
4.36-6.174.30.05416.124870.0290.0610.054100
6.17-53.5924.10.05615.714140.0320.0650.05699.8

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Processing

Software
NameVersionClassification
REFMAC5.8.0257refinement
iMOSFLMdata reduction
SCALAdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5N9O

5n9o


Resolution: 1.95→64.53 Å / Cor.coef. Fo:Fc: 0.912 / Cor.coef. Fo:Fc free: 0.897 / WRfactor Rfree: 0.2628 / WRfactor Rwork: 0.2188 / FOM work R set: 0.7786 / SU B: 4.36 / SU ML: 0.121 / SU R Cruickshank DPI: 0.1697 / SU Rfree: 0.1557 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.17 / ESU R Free: 0.156 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : REFINED INDIVIDUALLY
RfactorNum. reflection% reflectionSelection details
Rfree0.2508 2058 4.9 %RANDOM
Rwork0.2136 ---
obs0.2155 39743 98.78 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso max: 103.76 Å2 / Biso mean: 24.992 Å2 / Biso min: 11.52 Å2
Baniso -1Baniso -2Baniso -3
1-0 Å20 Å2-0 Å2
2--0.01 Å2-0 Å2
3----0.01 Å2
Refinement stepCycle: final / Resolution: 1.95→64.53 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3532 0 2 317 3851
Biso mean--63.71 36 -
Num. residues----452
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.010.0133632
X-RAY DIFFRACTIONr_bond_other_d0.0010.0173258
X-RAY DIFFRACTIONr_angle_refined_deg1.7831.6614928
X-RAY DIFFRACTIONr_angle_other_deg1.3491.5887563
X-RAY DIFFRACTIONr_dihedral_angle_1_deg7.7245452
X-RAY DIFFRACTIONr_dihedral_angle_2_deg32.4823.82178
X-RAY DIFFRACTIONr_dihedral_angle_3_deg13.75515586
X-RAY DIFFRACTIONr_dihedral_angle_4_deg16.2741512
X-RAY DIFFRACTIONr_chiral_restr0.0680.2466
X-RAY DIFFRACTIONr_gen_planes_refined0.0090.024079
X-RAY DIFFRACTIONr_gen_planes_other0.0030.02741
LS refinement shellResolution: 1.95→2.001 Å / Rfactor Rfree error: 0 / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.287 150 -
Rwork0.258 2943 -
all-3093 -
obs--100 %

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